Project Summary/Abstract The structure-function paradigm is a powerful guiding principle that underlies much of our understanding of biological processes. What is often neglected in this picture, however, is the flexibility of protein structures. This flexibility is necessary for a protein to fold to its native, active structure. Furthermore, protein function requires evolution of this native structure with time. Therefore, the dynamics of the protein structure and associated solvent water provide the critical connection between structure and function. The overall goal of this proposal is to elucidate the functional dynamics of hemagglutinin and M2 proton channel that enable influenza virus infection, a problem with significant public health implications. The mechanisms explored in this work are also relevant to other enveloped viruses, in particular HIV. More generally, membrane fusion and proton transport through proteins are of high fundamental interest and we expect the insight gained in these specific studies will contribute to the understanding of a broad range of related systems. We plan to pursue three specific aims: 1) Determine the mechanism of hemagglutinin mediated membrane fusion. We will test a new model for protein mediated membrane fusion that is based on molecular dynamics simulations of this process. We have developed unique methodology base on a laser induced pH jump to initiate the fusion process, and structure specific spectroscopic methods to characterize the hemagglutinin refolding dynamics that drive membrane fusion. This viral protein serves as an archetype for understanding the general mechanism of membrane fusion as a ubiquitous membrane transport process. 2) Determine the mechanism of fusion pore formation. We will test the hypothesis that the hemagglutinin trans-membrane domain (TMD) and fusion peptide (FP) form an oligomeric complex that opens and stabilizes the fusion pore. 3) Determine the molecular mechanism of actively gated proton transport. This work will on a focus on the influenza M2 proton channel, an important model ion channel. Understanding transport of protons through protein channels is critical to many essential biological processes as well as replication of the influenza virus. These aims are linked intellectually by energy landscape concepts and operationally by the methodology developed in our lab for studying both protein and membrane dynamics. Our unique approach will allow us to identify specific protein motions involved in protein mediated membrane fusion and proton channel activation. We expect this work to provide important new insight into the factors that shape the energy landscape of membrane proteins and the coupled membrane dynamics.